| The lotus(Nelumbo nucifera Gaertn.) seed contains health-promoting and nutritional substances. Lotus seed extracts have shown an antioxidant potential and free radical scavenging properties. However, there have been no reports about the assay of the antioxidant activity, anticancer activity and identification of flavonols from the fresh seed epicarp of N. nucifera (FSENN). The aim of this study was to examine the antioxidant potential of the fractions of extract (60% MeOH) of FSENN (EFSENN), the flavonols found in a fraction of an extract were identified, their levels were measured and their anticancer effect understood. Here, modern analysis techniques and biochemistry and molecular biology techniques were applied systematically to investigate the structure and bioactivities of flavonols from EFSENN. The main research contents and results are as follows.1. Extraction flavonols and fractionation of FSENNThe extraction of flavonols from FSENN by aqueous methanol (60%, v/v) yielded 25 g from 2000 g of seed epicarp of N. nucifera (1.25%). EFSENN (25 mg/mL) was fractionated by using a Toyopearl TSK HW-40s column, and three fractions were obtained, namely Fr1 (130 mg), Fr2 (1.8 g) and Fr3 (200 mg). The amount of total polyphenols in the EFSENN fractions was determined by the Folin-Ciocalteu method., Fr3 (872.87 mg gallic acid equivalents (GAE)/g) was found to be higher than Fr2 and Frl, which showed a phenolic content of about 768.11 and 138.75 mg GAE/g, respectively.2. Identification of flavonols and flavonol contents in Fr2 of EFSENNHPLC-UV was used to identify and determine the contents of aglycones in Fr2 of EFSENN. Four aglycones (myricetin, quercetin, kaempferol and isorhamnetin) were identified by HPLC retention times with corresponding standards. We determined the contents of aglycones by the calibration of the four standards (myricetin, quercetin, kaempferol and isorhamnetin). Our results indicated that the quercetin content of Fr2 (10.2 mg/g of dry fraction) was higher than isorhamnetin (6.2 mg/g of dry fraction), followed by kaempferol (2.8 mg/g of dry fraction) and myricetin (0.4 mg/g of dry fraction).3. LC/UV/MS analysisOn-line LC/UV/MS analysis was performed on a HPLC system with a UV photodiode array detector coupled to a mass spectrometer. MS data, especially in the negative mode, provided valuable information, including the molecular weight, information on the aglycone and sugar. Six glycosylated and one aglycone flavonol in Fr2 were found. Peak 1 was deduced as myricetin-monohexosides based on the folling information:the molecular ion [M-H]-at m/z 479, aglycone ion [A-H]-at m/z 316.9, fragment ion at m/z 150.9 from ring A and Other fragment ion at m/z 270.9. Peak 2 was confirmed as rutin (609 m/z) by co-elution with standard rutin. Peak 3 was identified as a quercetin monohexoside, the [M-H]-ion in ESI-MS spectrum was observed at m/z 463, while ESI-MS2 ion at at m/z 300.9. Peak 4 was deduced as kaempferol-monodeoxyhexoside-monohexoside based on the folling information:the [M-H]-ion at m/z 593 and [A-H]-ion at m/z 285. Peak 5 was identified as a isorhamnetin-monodeoxyhexoside-monohexoside, the [M-H]-ion at at m/z 623 whereas a [A-H]-ion at m/z 315 with Other fragment ions at m/z 270.9 and m/z 254.9. Peak 6 was deduced as isorhamnetin monohexoside based on [M-H]-ion at m/z 477, [A-H]-ion at m/z 315, fragment ion at m/z 150.8 from ring A and fragment ion at m/z 270.9. Peak 7 was finally confirmed as quercetin based on [M-H]-ion at m/z 301, whereas the other fragment ions at m/z 178.9, m/z 150.8 and m/z 106.9 were because of the fragmentation of rings A and 121.1 fragment from ring B of quercetin. 4. Antioxidant activity of fractions of EFSENNAntioxidant activity was evaluated in vitro by a number of methods including 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging, theβ-carotene bleaching method, ferric ion reducing capacity and total antioxidant capacity (T-AOC). The DNA damage-preventing effect of the fractions was determined by the CuSO4-Phen-Vc-H2O2-DNA CL system. The scavenging ability of the three fractions on hydroxide radicals (CuSCO4-Phen-Vc-H2O2 system) and hydrogen peroxide (luminol-H2O2 system) was investigated by using the chemiluminescence (CL) method in vitro. The result showed Fr3 had the highest DPPH scavenging activity. Fr3 exhibited the highest antioxidant activity followed by Fr2 and Fr1 in theβ-carotene bleaching assay. Our results showed that the fractions of seed epicarp have a remarkable potential to reduce ferric ions into ferrous ions. The T-AOC varied in different fractions ranging from 32.4 to 113.4 U/mg of the sample. Fr2 exhibited the highest T-AOC, followed by Fr3 and Fr1. The ability of preventing DNA damage of Fr3 was observed as higher than F2 and Fr1. The IC50 of Fr3 was 5±0.85μg/mL and 8.3±0.09μg/mL for Fr2, whereas Fr1 showed the highest IC50 value of 46.2±0.13μg/mL. Fr2 showed the lowest IC50 value (40±0.14μg/mL) followed by Fr3 and Frl in the scavenging ability of hydroxyl radical. Fr2 and Fr3 exhibited the same IC50 value (0.62μg/mL) in the scavenging effect of hydrogen peroxide.5. In vitro anticancer screeningThe anticancer activity of flavonols in Fr2 of EFSENN against HeLa cells was assessed by MTT assay, flow cytometry analysis and western blotting analysis. MTT assay showed the dose-dependent inhibitory effect on cell growth, the IC50 was 50μg/mL, and the maximal inhibition of cell growth (>90%) was obtained at 200μg/mL. The cells treated with 150μg/mL showed a progressive increase of 15.96±0.59% of the G2/M phase. The increase in G2/M phase cells after Fr2 treatment was accompanied by a decrease in G1 populations relative to control cultures. These results suggested that flavonols in Fr2 inhibited cellular proliferation via G2/M phase arrest in a dose-dependent manner. Furthermore, the up-regulation of Bax, down-regulation of Bcl-2 and inhibition of NF-κB were observed by western blotting analysis.6. ConclusionThe fractions of EFSENN possess a high antioxidant potential and Fr2 showed high levels of flavonol compounds with an anticancer effect, which are beneficial for the treatment or prevention of a variety of diseases and have a nutraceutical potential. |
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